Inhalation anesthesia induced by isoflurane alters penicillin disposition in swine tissues.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24 h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28 h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8 h postdose, and a second set of four similarly treated control pigs were sacrificed 28 h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

Tissue/fluid correlation study for the depletion of sulfadimethoxine in bovine kidney, liver, plasma, urine, and oral fluid.

Sulfonamides are among the oldest, but still effective, antimicrobial veterinary medicines. In steers and dairy cows, the sulfonamides are effective in the treatment of respiratory disease and general infections. Sulfadimethoxine (SDM) has been approved by US Food and Drug Administration (FDA) for use in steers and dairy cows with a tolerance of 100 ng/g (ppb) in edible tissues and 10 ppb in milk. The detection of SDM residue above tolerance in the animal slaughtered for food process will result in the whole carcass being discarded. This report describes a comprehensive depletion study of SDM (and its main metabolite) in plasma, urine, oral fluid, kidney, and liver. In this study, nine steers were injected intravenously with the approved dose of SDM; the loading dose was 55 mg/kg, followed by 27.5 mg/kg dose at 24 h and again at 48 h. Fluids (blood, urine, and saliva) and tissue (liver and kidney) samples were collected at intervals after the last dose of SMD. The combination of laparoscopic serial sampling technique with the liquid chromatography/mass spectrometry method provided the data to establish the tissue/fluid correlation in the depletion of SMD. A strong correlation and linearity of the log-scale concentration over time in the depletion stage has been confirmed for kidney, liver, and plasma.

Confirmation of gentamicin and neomycin in milk by weak cation-exchange extraction and electrospray ionization/ion trap tandem mass spectrometry.

A new procedure for the confirmation of two aminoglycoside antibiotics in milk was developed and validated. This work is among the early applications of ion trap mass spectrometry for regulatory methodology, and it incorporates a novel weak cation-exchange extraction. The procedure was validated for the confirmation of both gentamicin and neomycin at 30 ng ml(-1) and above. Milk is first treated with acid and centrifuged. The supernate, excluding the fat layer, is buffered with sodium citrate to neutral pH. The extract is applied to a weak cation-exchange solid-phase extraction column. Aminoglycosides are eluted with acidified methanol. Following separation by ion-pair liquid chromatography, analytes are ionized with an electrospray interface. Protonated molecular ions are selectively stored in an ion trap mass spectrometer, then collisionally dissociated to yield unique product ion spectra. Confirmation is based on matching spectral responses between samples and comparison standards consisting of a bona fide standard spiked into control extracts. Method performance was demonstrated with replicate samples of control milk, fortified milk, and milk containing incurred residues of each compound.

Time preference in medical decision making and cost-effectiveness analysis.

Cost-effectiveness analyses usually quantify peoples' attitudes towards delayed outcomes using the exponential discount model. The authors examined three assumptions of this model by assessing the time preferences of individuals towards hypothetical health states and calculating implicit annual discount rates. Of a random sample of medical students, house officers, and attending physicians, 121 participated, reflecting a response rate of 81%. The participants considered three temporary events (colostomy, blindness, depression) that were destined to occur at five sequentially distant times in the future (one day, six months, one year, five years, and ten years). The utility of each prospect was measured using two elicitation techniques (standard gamble and categorical scaling), and 1,394 implicit discount rates were calculated. Of all the discount rates, 62.1% equalled zero, 10.0% were less than 0.00, and 15.7% were greater than 0.10. Mean discount rates for relatively proximal time intervals tended to be larger than those for relatively more distant intervals (0.041 vs. 0.025, p < 0.01). Mean discount rates for blindness tended to be smaller than those for colostomy or depression (0.023 vs. 0.039 vs 0.037, respectively, p < 0.005). Hence, peoples' implicit discount rates are not always small positive numbers that are constant over time and the same for all settings. The authors suggest that the conventional exponential discount model may not fully characterize the time preferences held by individuals.

Method for determination of pentobarbital in dry dog food by gas chromatography/mass spectrometry.

A procedure has been developed and validated for measuring the concentration of pentobarbital residues in dry, extruded animal feed in the range of 3-200 ng/g (ppb) with an estimated limit of quantitation of 2 ppb. The method was developed for surveillance purposes: to measure the concentration of euthanizing agent which might be present in feeds incorporating rendered products which themselves might include some fraction of euthanized animals. A previously published qualitative procedure was modified by adding isotopically labelled pentobarbital as an internal standard. Dry feed was ground and extracted with methanol. The extract was loaded on a mixed-mode (C-18, anion exchange) solid-phase extraction cartridge designed for barbiturate residues. Pentobarbital was eluted and derivatized for gas chromatography/mass spectrometry in positive ion chemical ionization mode. Quantitation was based on the ratio of dimethyl-pentobarbital MH+ (m/z 255) vs dimethyl-pentobarbital-d(5) (m/z 260) in standards and extracts. Accuracy ranged from 112% at 3 ppb to 96% at 200 ppb, with relative standard deviations ranging from 4% at 3 ppb to 2% at 200 ppb.

Identification of cephapirin metabolites and degradants in bovine milk by electrospray ionization--ion trap tandem mass spectrometry.

Liquid chromatography-ion trap tandem mass spectrometry (LC-MS/MS) with electrospray ionization was used to identify cephapirin metabolites and degradants in milk from cows dosed with cephapirin. The milk was extracted according to a previously published procedure. Structures for various components were tentatively identified by their molecular weight, product ion mass spectra, and/or correspondence to standard mass spectra. These components may have occurred as metabolites or as degradants that occurred on storage or during extraction. Compounds identified in the milk included cephapirin, desacetylcephapirin, cephapirin lactone, hydrolyzed cephapirin, and a reduced cephapirin lactone that has not previously been reported. Methylcephapirin was also identified, possibly as a trace contaminant in the formulation. Analysis of incurred milk extracts showed that cephapirin and desacetylcephapirin are the major residues in milk. Desacetylcephapirin residues persisted about as long as the parent drug. The detection limit for both residues by LC-MS/MS was approximately 1 ng/mL in milk. These results have implications for microbiological methods or rapid test kits, if such methods or kits respond to cephapirin metabolites and degradants present in the milk.

Determination and confirmation of pirlimycin residue in bovine milk and liver by liquid chromatography/thermospray mass spectrometry: interlaboratory study.

Three laboratories participated in trials of methods to determine and confirm pirlimycin residue in bovine milk and liver. The methods used liquid chromatography/thermospray mass spectrometry (LC/MS) with an internal standard to measure residue concentration. The internal standard was isopirlimycin, a stereoisomer of pirlimycin, which was resolved chromatographically. Determinative procedures were validated by replicate analyses of negative control, fortified control, and residue-incurred milk. For the milk method, average corrected recoveries (and coefficients of variation, CVs) were 83-113% (CV, 7.5-15.4%) at 0.2 ppm, 91-98% (CV, 3.4-18.5%) at 0.4 ppm, and 89-102% (CV, 8.8-22.9%) at 0.8 ppm. For the liver method, average corrected recoveries were 94-103% (CV, 2.2-7.1%) at 0.25 ppm, 87-94% (CV, 4.8-10.3%) at 0.5 ppm, and 96-101% (CV, 5.5-6.9%) at 1.0 ppm. There were no interferences in control samples of either matrix. Pirlimycin was confirmed by matching the retention time and relative abundances of 4 ions from sample extracts to corresponding values obtained for pirlimycin standard. Pirlimycin was confirmed in all residue-incurred samples and all samples fortified at regulatory tolerances (0.4 ppm in milk and 0.5 ppm in liver) by 2 of the 3 laboratories and in most samples by the third laboratory.

Determination of pirlimycin residue in milk by liquid chromatographic analysis of the 9-fluorenylmethyl chloroformate derivative.

A procedure to determine pirlimycin residues in bovine milk was developed. Pirlimycin was extracted from milk after protein precipitation and 2 stages of liquid-liquid partitioning. The extract was evaporated to dryness, redissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (Fmoc) to impart a chromophore for ultraviolet (UV) detection. The derivatized extract was analyzed by reversed-phase liquid chromatography (LC). Pirlimycin concentration was calculated from the peak height of the extract by using a linear regression standard curve prepared from a series of derivatized standards. The procedure's performance in the range of the regulatory tolerance (0.4 ppm) was validated by replicate analysis of control, fortified control, and residue-incurred bovine milk. Average recoveries at 0.2, 0.4, and 0.8 ppm were 91, 88, and 87%, respectively, and coefficients of variation were 5, 4, and 1%, respectively. Overall recovery for all samples was 89%, with 4% coefficient of variation. Linear regression analysis of LC/UV results versus results from a previously published LC/mass spectrometric assay gave a slope of 0.95, with a correlation coefficient of 0.98.

Confirmation of leucogentian violet in chicken fat by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric (GC/MS) procedure for confirming the identity of leucogentian violet (LGV) in chicken fat was developed for regulatory application. The unused portion of the extract remaining from a determinative procedure was back-extracted into an organic phase, concentrated, and analyzed by GC/MS. Confirmation of the identity of LGV was based on matching the retention times and relative abundances of 6 ions in the extract to corresponding values obtained for the LGV standard. The procedure was validated by replicate analyses of negative control, fortified control, and residue-incurred chicken fat. The presence of LGV was confirmed by the GC/MS procedure in all samples found to contain LGV by prior liquid chromatographic analyses. There were no interferences in the control samples.

Liquid chromatography/mass spectrometry for timely response in regulatory analyses: identification of pentobarbital in dog food.

A limited liquid chromatography/mass spectrometry (LC/ MS) data set was acquired under conditions which called for timely response without benefit of a fully developed method. Quality assurance elements verified that an LC/ MS procedure developed in a short-time was sufficiently under control to meet its purpose. LC/MS was used to rule out a potential problem with a gas chromatography (GC)/MS method that had been developed for regulatory purposes. The LC/MS data set showed that signals identified by GC/MS as diagnostic of pentobarbital (PB) were not artifacts of derivatization or GC analysis. Samples of dry dog food identified by GC/MS as containing PB were also shown by LC/MS to contain PB. The LC/MS method would not be recommended as a substitute for GC/MS, primarily because of poorer sensitivity. Although the data set is limited, and justifiably represents only the starting point for conventional method development, the purpose at hand was served adequately. This work demonstrates the utility of LC/MS for rapid regulatory response, provided there is a framework of quality assurance checks.

Particle beam liquid chromatography/mass spectrometry with negative ion chemical ionization for the confirmation of ivermectin residue in bovine milk and liver.

Particle beam liquid chromatography/mass spectrometry (LC/MS) using negative ion chemical ionization was applied to the analysis of ivermectin residue in bovine milk and liver. Samples were prepared by liquid/liquid extraction followed by alumina B solid-phase extraction clean-up. On-line LC/MS of extracts was carried out on a C-18 bonded silica column. Signals were observed from on-column injections of 4 ng dihydro-avermectin B1a (H2B1a) in extracts equivalent to 2 ml milk or 0.3 g liver. The specificity required for a regulatory confirmation procedure was achieved by monitoring the H2B1a molecular ion and four fragment ions. Ion chromatogram peak areas were at least three times greater than control samples integrated over the same time window. Coeluting matrix compounds enhanced the response and altered the abundance pattern of H2B1a. To compensate for this matrix effect, control milk extracts were spiked with H2B1a standard and used for the abundance matching for the abundance matching requirement of regulatory confirmation.

Confirmation of spectinomycin in milk using ion-pair solid-phase extraction and liquid chromatography-electrospray ion trap mass spectrometry.

A confirmation procedure is described for residues of spectinomycin in bovine milk. Spectinomycin is extracted from raw milk using ion-pair reversed-phase solid-phase extraction. The extracts are ion-pair chromatographed on a polymeric reversed-phase column and analyzed on a quadrupole ion trap mass spectrometer equipped with an electrospray interface. MS-MS data are acquired in the scan mode of product ions deriving from m/z 333, the protonated molecular ion. The estimated limit of confirmation is between 0.05 and 0.1 microg/ml. The procedure was validated with control milk, fortified milk (0.1-5.0 microg/ml), and milk from cows dosed with spectinomycin.

Electrospray ionization and tandem ion trap mass spectrometry for the confirmation of seven beta-lactam antibiotics in bovine milk.

The feasibility of a technique to confirm the presence of residues from seven beta-lactam antibiotics in bovine milk has been demonstrated. The technique makes use of electrospray ionization and tandem ion trap mass spectrometry. Residues are first extracted from milk by reversed-phase solid phase extraction. Target analytes are separated by on-line reversed-phase liquid chromatography and ionized in the electrospray interface. The product ion mass spectra are acquired following collision-induced dissociation of protonated molecules. Confirmation is based on comparison of full scan spectra between unknowns and bona fide standards. The feasibility of this technique has been demonstrated for the six beta-lactams currently approved for use in lactating dairy cattle (penicillin G, ampicillin, amoxicillin, cloxacillin, cephapirin and ceftiofur) and a drug not approved for animal use, cefazolin. The technique has been applied to control milk fortified at 5 ng/mL of penicillin G and 10 ng/mL of the other six drugs.

Determination of the sialylation pattern of human fibrinogen glycopeptides with fast atom bombardment.

Sialylated biantennary glycopeptides from human fibrinogen were analyzed with fast atom bombardment mass spectrometry. The mass spectrometric conditions used in the positive mode showed predominantly molecular ions with no fragment ions due to the loss of sialic acid. Standard mixtures of glycopeptides with zero, one, and two sialic acid residues revealed a linear relationship between ion abundance and molar fraction. The desorption efficiency varied according to the number of sialic acid residues in these biantennary glycopeptides. The relative abundance of different molecular ion species differing only in amino acid content was in agreement with chemical analysis. Sensitivity, precision, and requirements for sample preparation were assessed. Both assignment of molecular weights and quantification of individual glycopeptide species from human fibrinogen were obtained. The glycopeptides from human fibrinogen were found to consist of a mixture of equal amounts of monosialylated and disialylated species with no asialoglycopeptides.

Mass spectrometric studies of a modified active-site tetrapeptide from delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni.

Examination of the product of affinity labeling of delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni by the suicide substrate [7-3H]5,10-secoestr-5-yne-3,10,17-trione has demonstrated that the steroid becomes bound by an acid- and base-labile linkage to an active-site peptide representing residues 55-58 (H2N-Tyr-Ala-Asn-Ser-CO2H) of the primary structure (Penning, T. M., and Talalay, P. (1981) J. Biol. Chem. 256, 6851-6858). Upon release of the steroid by mild acid hydrolysis, the peptide is converted into a more basic structure while retaining its amino acid composition (as determined by conventional means). These findings were rationalized by postulating that the steroid is bound as an imido ester via the amide group of asparagine 57 and that the polypeptide backbone participates (via attack by nitrogen or oxygen) in the hydrolysis of this ester with the formation of a cyclic amidine or basic oxazine. By comparing the properties of the isolated tetrapeptide, from which the steroid has been removed, with those of synthetic H2N-Tyr-Ala-Asn-Ser-CO2H and H2N-Tyr-Ala-Asp-Ser-CO2H by electron impact and fast atom bombardment mass spectrometry, we now have evidence for the presence of an oxazine (5,6-dihydro-6-iminio-4H-1,3-oxazine) in the modified peptide. Our results draw attention to the hitherto unsuspected degree of nucleophilicity of the amide group of the side chain of asparagine and the participation of this group in the formation of an imido ester.

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry.

The capability of fast atom bombardment mass spectrometry for characterization of phosphorylation sites in a tryptic peptide from chicken egg yolk riboflavin-binding protein has been evaluated. The quality of information about molecular weight, amino acid sequence, phosphorylation sites, and microheterogeneity is evaluated as a function of the sign of the ions analyzed, the nature of the counter ions associated with the phosphate substituents, sample matrix, and various instrumental parameters. The intact octaphosphorylated 23-residue peptide was found to be susceptible to mass spectral analysis. Information from the negative ion spectrum was used in conjunction with complete sequence information and experiments which showed that all phosphates were attached to serine residues. Phosphorylated and unphosphorylated serine residues were identified and the sample was shown to be homogeneously octaphosphorylated.

Desorption of ions from rat membranes: selectivity of different ionization techniques.

Complex lipid biomarkers, including phosphatidylcholines, cerebrosides and sulfatides, are shown to be desorbed intact from rat brain myelin and rat liver microsomes by liquid secondary ion mass spectrometry, by plasma desorption and by laser desorption. Different polar lipids are favored by the different desorption techniques and as negative or positive ions. These selectivities support current theories about ionization for the different techniques.

Mass spectral analysis of complex lipids desorbed directly from lyophilized membranes and cells.

Three desorption ionization techniques--laser desorption, plasma desorption and fast atom bombardment mass spectrometry--have been applied to lyophilized cells, membranes, lysed cells and various extracts. It has been shown that intact polar lipids are selectively desorbed from biological membranes by these methods and that their mass spectra provide "fingerprints" which reflect the unique biochemical composition of each class of cell or membrane.